1. Field of the Invention
The invention provides apparatus and methods for holding and circulating fluids over a membrane. The invention may find particular use in laboratory studies of the transport and absorption of substances by membranes including biological tissues, cell cultures, and synthetic materials. The invention may also find use in culturing cells in vitro.
2. Description of the Background Art
Hobson, U.S. Pat. No. 4,667,504, issued May 26, 1987, discloses an apparatus for determining in vitro the penetration rate of chemicals across a biological membrane. The apparatus comprises two housings, one holding a reservoir of test chemical, and the other providing a chamber for flowing receptor solution across a membrane held in a membrane holding compartment. The membrane holding compartment comprises a cylindrical depression surrounding an open end of the receptor solution chamber. The receptor solution chamber is tilted slightly with its higher end open to the membrane holding compartment. The tilt prevents bubbles in the receptor solution from becoming stalled or trapped inside the chamber and interfering with the reliability and reproducibility of tests. An inlet bore from the upper surface of the receptor housing leads to a closed end of the chamber near the membrane holding compartment to the upper surface of the receptor housing. The inlet bore is sized smaller than the outlet bore. The depth of the depression forming the membrane holding compartment varies, tapering from a lesser depth at its intersection with the chamber to a greater depth at the outer circumference of the depression. The thus formed truncated cone ensures that a sample biological membrane is stretched taut over the chamber opening by the force fastening the reservoir housing to the receptor solution housing.
The publication of Ussing and Zerahn (Acta Physiol. Scand. 23:110-127 (1951)) describes an apparatus for the determination of sodium flux and short circuit current in frog skin. The device has an area for placement of the skin between two opposing half cells, pressed against the skin by two lucite screws held by steel uprights. The tips of the lucite screws are conical and fit into conical depressions in the center of the ebonite dishes. Buffer solutions are circulated and aerated by air entering through side tubes. Bridges, held in position by pieces of rubber tubing, fit tightly into short celluloid side tubes sealed into the two chambers and these connect to a reservoir system. The device is also fitted with a series of electrodes.
Schoenwald and Huang (J. Pharm. Sci. 72:11 (1983)) discloses a device for mounting of corneas to determine transport of material across corneal tissue. The system is composed of two acrylic plastic blocks. Each block acts as an opposing half cell with an area for the corneal tissue between. The cornea is mounted using a system of three rings, and the ring system with the cornea is positioned in the opening of the blocks and forms a water-tight seal when sufficient lateral pressure is applied to the blocks in a manner similar to that described above by Ussing and Zerahn. Fluid reservoirs on each side of the tissue are mixed with a gas lift mechanism, also similar to Ussing and Zerahn above. Stirring motors mounted on the blocks connect by shafts with blades on the terminus through the block to the reservoir at the tissue face. These blades promote mixing at the tissue surface. The reservoirs are heated by circulation of temperature controlled fluid through channels in the block. The fluid reservoirs and fluid circulation channels for temperature control are within the same contiguous block.
Hidalgo et al. (Gastroenterology 96:736-49 (1989)) disclose a method for the measurement of transport of material across a monolayer formed by cultured cells. Cells are grown in an appropriate medium in a plastic cylinder which is capped at one end with a filter membrane. When the cells reach a confluent monolayer, the cells and the cylinder with attached filter are placed in another larger diameter cylinder with fluid which acts as the receiver solution. Materials placed in the smaller cylinder with the cells are transported across the cells and the filter membrane to the reservoir solution of the larger cylinder.
Grass and Sweetana (Pharm. Res. 5:6 (1988)) disclose a diffusion cell chamber for the measurement of tissue permeability. The apparatus is comprised of at least one chamber with a first and a second volume element, each of which contains a reservoir for fluids and a means to circulate fluids. Also included is a means to retain a membrane separating the first and second volume elements whereby the fluid contained in the reservoir in the first volume element housing could communicate with the fluid contained in the reservoir in the second volume element in the absence of the membrane. Also included are a membrane separating the first and second volume elements, a means to circulate fluid contained in each of the first and second volume elements, and a means to attach the first and second volume elements on a contiguous surface of each of the first and second volume elements in an adjacent facing relationship, wherein the first and second volume element housings are separated from each other at their contiguous surfaces by the membrane. Substantially the same apparatus is disclosed in Sweetana et al, U.S. Pat. No. 5,183,760, issued May 26, 1987.
Co-pending U.S. patent application Ser. No. 08/138,653 (attorney docket no. 14664-2), filed Oct. 8, 1993, now abandoned in favor of U.S. application Ser. No. 08/506,049, filed Jul. 24, 1995, discloses a two chamber membrane holder in which a membrane held between a pair of housings divides the holder into two discrete volumes in which first and second fluids are held and through which those fluids may be circulated.
The disclosure of each of the above publications is hereby incorporated herein by reference.